Transcriptome-wide characterization, evolutionary analysis, and expression pattern analysis of the NF-Y transcription factor gene family and salt stress response in Panax ginseng

Jilin ginseng (Panax ginseng C. A. Meyer) has a long history of medicinal use worldwide. The quality of ginseng is governed by a variety of internal and external factors. Nuclear factor Y (NF-Y), an important transcription factor in eukaryotes, plays a crucial role in the plant response to abiotic stresses by binding to a specific promoter, the CCAAT box. However, the NF-Y gene family has not been reported in Panax ginseng. In this study, 115 PgNF-Y transcripts with 40 gene IDs were identified from the Jilin ginseng transcriptome database. These genes were classified into the PgNF-YA (13), PgNF-YB (14), and PgNF-YC (13) subgroups according to their subunit types, and their nucleotide sequence lengths, structural domain information, and amino acid sequence lengths were analyzed. The phylogenetic analysis showed that the 79 PgNF-Y transcripts with complete ORFs were divided into three subfamilies, NF-YA, NF-YB, and NF-YC. PgNF-Y was annotated to eight subclasses under three major functions (BP, MF, and CC) by GO annotation, indicating that these transcripts perform different functions in ginseng growth and development. Expression pattern analysis of the roots of 42 farm cultivars, 14 different tissues of 4-year-old ginseng plants, and the roots of 4 different-ages of ginseng plants showed that PgNF-Y gene expression differed across lineages and had spatiotemporal specificity. Coexpression network analysis showed that PgNF-Ys acted synergistically with each other in Jilin ginseng. In addition, the analysis of the response of PgNF-YB09, PgNF-YC02, and PgNF-YC07-04 genes to salt stress treatment was investigated by fluorescence quantitative PCR. The expression of these genes increased after salt stress treatment, indicating that they may be involved in the regulation of the response to salt stresses in ginseng. These results provide important functional genetic resources for the improvement and gene breeding of ginseng in the future. Conclusions: This study fills a knowledge gap regarding the NF-Y gene family in ginseng, provides systematic theoretical support for subsequent research on PgNF-Y genes, and provides data resources for resistance to salt stress in ginseng. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-022-03687-6.

transcription factors with histidine-like subunits that are uniquely characterized by their binding to DNA at the CCAAT site as a heterotrimeric complex consisting of NF-YA, NF-YB and NF-YC, three individual subunits of the protein family [2]. To date, members of the NF-Y gene family have been identified in a variety of plants, including Arabidopsis thaliana [3], Oryza sativa [4], Prunus persica [5], and Populus [6]. Studies have shown that the NF-Y gene family influences flowering in plants [7], improves photosynthetic capacity [8], regulates embryogenesis in Arabidopsis [9], and assists in abiotic stress resistance [10][11][12]. NF-Y family members are involved in plant responses to abiotic stresses, and ZmNF-YB16 functions by regulating the expression of photosynthesisrelated genes to improve the antioxidant capacity of cells and thus achieve drought resistance [13]. In Lycopersicon esculentum, SlNFYA10 negatively regulates the AsA (ascorbic acid) biosynthetic pathway by binding to the CCAAT box in response to oxidative stress [14]. Overexpression of the GmNFYA5 gene in soybean enhances drought resistance [15]. Although the NF-Y gene family has been extensively studied in other species, it has not been reported in ginseng.
Jilin ginseng (Panax ginseng C.A. Meyer) is a perennial herb in the Araliaceae family that has a cultivation history of at least 4,000 years. The Chinese "Sheng Nong's Herbal Classic" records in detail that ginseng tastes sweet, can be used as a tonic for the five organs, calms the spirit, fixes the soul, stops panic and palpitations, removes evil spirits, brightens the eyes, makes the mind happy, educates the mind, lightens the body and prolongs life when taken for a long time [16]. Ginseng is not currently used by consumers as a single medicinal plant but as a food and health product [17]. The quantity of wild ginseng can hardly meet the social demand for ginseng products, while the quality of artificially cultivated ginseng is constrained by various factors. Some members of the NF-Y gene family respond to salt stress. We obtained the NF-Y gene family members in response to salt stress treatment by screening the adventitious roots of ginseng, which will provide genetic resources for the subsequent improvement of salt stress resistance in ginseng cultivars.
In this study, 40 PgNF-Y genes were identified in the Jilin ginseng transcriptome database and classified according to their structural domain information (NF-YA, NF-YB, and NF-YC). Subsequently, we analyzed the evolutionary relationships of the PgNF-Y gene family, and conserved motifs and annotated them with GO functions. In addition, expression pattern analysis and coexpression network analysis were performed based on the PgNF-Y gene expression data. Finally, the response of PgNF-Y family members to different concentrations of salt was explored. The results of this experiment provide important theoretical information and experimental data on the NF-Y gene family for the subsequent study of functional genes in ginseng.

Identification of the NF-Y genes in Panax ginseng
To maximize data integrity and reliability, we adopted three different approaches to screen members of the NF-Y gene family in the Jilin ginseng database containing 248,993 transcripts [18]. First, the hidden Markov model of the NF-Y gene family was downloaded from the PFAM protein family database (http:// pfam. xfam. org/), and using PFAM IDs (PF02045, PF00808) as the interrogated sequences, tBlastn was performed in the Jilin ginseng transcriptome. Second, the coding and protein sequences of NF-YA, NF-YB, and NF-YC were downloaded from the Korean Ginseng Genome website (http:// ginse ngdb. snu. ac. kr/ pathw ay. php); these sequences were used as the interrogation sequences for Blastn and tBlast of the Jilin ginseng transcriptome database at an e-value of 1 × 10 -6 . Finally, 10 protein sequences from the NF-Y family with verified functions were downloaded from GenBank (https:// www. ncbi. nlm. nih. gov/) and used as interrogation sequences for tBlastn of the transcriptome of Jilin ginseng. These 10 sequences were from Arabidopsis thaliana [10,19], Triticum aestivum [20,21], Oryza sativa [22], Glycine max [19], Solanum tuberosum [8], Nicotiana tabacum [23], and Zea mays [11]. Subsequently, the results obtained by the three methods were combined, and after removing duplicate values, the files were used for our preliminary investigation. To exclude some spurious comparison information, we submitted the results to iTAK (http:// itak. feilab. net/ cgi-bin/ itak/ index. cgi) for online analysis while maintaining the sequence information regarding NF-Y structural domains. Based on the results of iTAK, all the obtained NF-Y transcripts were named PgNF-Ys. Finally, the obtained transcripts were verified one by one for structural domains by the SMART online tool (http:// smart. embl-heide lberg. de/).

Phylogenetic evolutionary analysis and conserved motifs of the PgNF-Y gene family in Panax ginseng
We identified 79 transcript sequences with complete open reading frames from 115 PgNF-Y gene transcripts based on ORF Finder in NCBI (the remaining 36 transcripts contained only the structural domain information of NF-Y). Based on these results, 12 nucleic acid sequences in the NF-YA, NF-YB, and NF-YC subgroups from Lycopersicon esculentum, Arabidopsis thaliana, Oryza sativa, and Helianthus annuus were downloaded from the NCBI database as outgroups for the phylogenetic tree. These nucleic acid sequences were translated into protein sequences. In MEGA-X software [24], the maximum-likelihood (ML) method was chosen to obtain the evolutionary tree of the PgNF-Y genes with 1,000 bootstrap replicates to further illustrate the evolutionary relationships between the PgNF-Y genes in different species. Finally, the evolutionary tree was optimized by Evolview (https:// www. evolg enius. info/ evolv iew/#/ treev iew). To determine the conserved sequence patterns in the NF-Y gene family members in Jilin ginseng, we analyzed the motifs of PgNF-Y transcription factors by the MEME online tool (https:// meme-suite. org/ meme/ doc/ cite. html? man_ type= web). Finally, the obtained results were visualized by TBtools [25].

GO (Gene Ontology) annotation and functional categorization of PgNF-Y gene transcripts
We used Blast2GO version 6.0.3 [26] to annotate the identified NF-Y transcripts based on Gene Ontology (GO) functional annotation into three major categories: BP (Biological Process), CC (Cellular Component), and MF (Molecular Function). The enrichment of all the nodes where the genes were located was determined by the Chi-square test at Level 2. The functions of 109,781 transcripts that were annotated by previous authors were used as reference information [18].

Expression pattern and network analysis of PgNF-Y gene transcripts
Since gene expression is subject to a variety of conditions, we obtained data on the expression of PgNF-Ys in 42 farm cultivars, 14 different tissues of 4-year-old ginseng, and 4-year-old ginseng roots, and we plotted the heatmap in R. Thus, the expression pattern of the PgNF-Y genes in Jilin ginseng in time and space was determined. To further investigate the interrelationship between PgNF-Y gene expression in 42 farm cultivars, Spearman's correlation coefficients of PgNF-Y gene expression were calculated using R, and BioLayout Express 3D version 3.3 software was used to form a visual network of the obtained results.

Analysis of the response of PgNF-Y genes to salt stress
In this experiment, the adventitious roots of Jilin ginseng (Panax ginseng C.A. Meyer) were treated with different concentrations of salt in B5 medium (0, 70, 80, 90, and 100 mM NaCl), and the treated adventitious roots were incubated under dark conditions at 22 °C for 30 days.

Plant materials, RNA isolation, and quantitative real-time PCR analysis
Adventitious root material (0.1 g) treated with different salt concentrations was weighed separately, and total RNA was extracted from ginseng adventitious root tissue using TRIzol (BioTeke, Beijing, China). The HiFiScript gDNA Removal cDNA Synthesis Kit (CWBIO, Beijing, China) was used to reverse transcribe the extracted RNA into cDNA. Actin 1 was selected as the internal reference gene, and according to the instructions for the UltraSYBR One-step RT-qPCR Kit (Low ROX) (CWBIO, Beijing, China), a 7500 Real Time PCR System was used to perform the reaction. The total reaction system was 10 µL, which included UltraSYBR Mixture (Low ROX) at 5 μL, upstream and downstream primers at 0.2 μL each, template at 1 μL, and ddH 2 O at 3.6 μL. qPCR was performed in a thermal cycling system with the following conditions: 95 °C for 10 min and 40 cycles of 95 °C for 15 s, and 60 °C for 60 s. To ensure the accuracy of the results obtained for each treatment, we set up 3 biological replicates and 3 technical replicates. The final results were obtained using the 2 −ΔΔCt analysis method.

Identification of PgNF-Y gene family transcripts
A total of 2,266 transcripts remained after removing duplicates from the NF-Y gene family transcripts obtained by three different methods for interrogating the Jilin ginseng transcriptome database. These transcripts were submitted to iTAK and queried for structural domains by SMART. Finally, 115 transcripts containing the structural domain of the NF-Y gene family were obtained. We collected basic information on the PgNF-Y gene family members of Jilin ginseng, including transcript ID, gene ID, mRNA sequence information, the number of transcripts with a length ranging from 240-2,624, and the number of amino acids with complete open reading frames (ORFs) ranging from 70-336 (Table S1).

Motif prediction and phylogenetic analysis of PgNF-Y genes
Since ginseng originated approximately 100 million years ago in the Cretaceous period, it has a long evolutionary history. We further investigated the evolutionary characteristics of PgNF-Y gene family members in Jilin ginseng based on 79 NF-Y gene family members with complete ORFs (the remaining 36 transcripts contained only structural domain information of NF-Y). We explored the evolutionary relationships of the PgNF-Y gene family from the perspectives of closely related species, model plants, monocotyledons, and dicotyledons. We selected Solanum lycopersicum, Arabidopsis thaliana, Oryza sativa, and Helianthus annuus as outgroups for phylogenetic analysis via the maximum likelihood method (Table S2). Phylogenetic tree analysis showed that the transcripts of the PgNF-YA, PgNF-YB, and PgNF-YC subgroups were all concentrated in specific subclades (except PgNF-YC04 and PgNF-YC10), and the NF-Y members that were distributed in the same subclass had functional similarity. Based on the evolutionary relationship between outgroups and PgNF-Y transcripts, NF-Y members in these species have an evolutionary origin from a common ancestor (Fig. 1A).
To understand the sequence characteristics of the PgNF-Y protein, its conserved structural domains were analyzed by the online tool MEME. The results showed that among the 10 motifs analyzed, the number of motifs contained in different subfamily members ranged from 1 to 5 (Fig. 1B). Motif 3, motif 6, and motif 7 were only present in the PgNF-YA subfamily; motif 4, motif 5, and motif 8 were only present in the NF-YC subfamily; and motif 2, motif 9, and motif 10 were commonly found in the NF-YB subfamily. The differences in the number and type of motif in different subfamilies demonstrate that the NF-Y gene family is functionally diverse and structurally different.

Expression characteristics of the PgNF-Y gene transcripts
To investigate the expression pattern of the PgNF-Y gene family from a temporal and spatial perspective, we retrieved PgNF-Y gene expression data from 4-year-old Jilin ginseng Damaya roots from the database containing 42 farm cultivars (S1-S42) and 14 different tissues (stem, fiber root, fruit peduncle, main root epiderm, fruit pedicel, rhizome, leaf peduncle, arm root, leaflet pedicel, leg root, leaf blade, fruit flesh, main root cortex, and seed), as well as four ginseng root samples of various ages (5, 12, 18, and 25 years) of PgNF-Y gene expression data (Table S4).
The results of the heatmap of the roots of 42 farm cultivars showed that 96 PgNF-Y transcripts (85%) were expressed in at least one cultivar and 36 transcripts (31%) were expressed in all 42 cultivars (Fig. 3C).
To further understand the expression trends of the PgNF-Y genes in Jilin ginseng, the expression of PgNF-Y transcripts was analyzed in the roots of 42 farm cultivars, 14 different tissues, and the roots of plants at 4 different ages. In the bar graphs showing the results for the roots of plants at four different ages (Fig. 4A), PgNF-Y tended to be more highly expressed in 5-year-old ginseng roots and less expressed in 12-year-old and 18-year-old ginseng roots. In the bar graph of the 14 different tissues within ginseng (Fig. 4B), the PgNF-Y transcripts tended to be expressed mostly in the fruit pedicel, while the leaf blade showed the lowest amount of PgNF-Y expression. Among the 42 farm cultivars of ginseng, the number of PgNF-Y transcripts expressed accounted for 53.04%-63.48% of the total PgNF-Y transcripts. To better understand the general level of PgNF-Y gene expression in 14 different tissues and 4 different ages, violin plots of gene expression versus tissue and age were plotted ( Fig. 5A and B). The results showed that PgNF-Y gene expression was lower in the main root cortex and leaf blade and higher in the fruit peduncle and fruit pedicel; the expression of PgNF-Y genes was relatively consistent across the four different ages. Based on this analysis of the expression pattern of PgNF-Y transcripts, we found that the expression of PgNF-Y genes in Jilin ginseng was influenced by geographical factors, tissues and plant age.

Coexpression network of PgNF-Y gene transcripts
To investigate whether there is a relationship between PgNF-Y transcripts among different genotypes, we performed a coexpression network analysis of PgNF-Y transcripts from 42 farm cultivars. We selected 96 of these transcripts for coexpression network analysis (the remaining 19 transcripts were not expressed in any of the 42 farm cultivars). The coexpression network results showed that at P ≤ 5.0E-02, the 96 transcripts formed a coexpression network containing 95 nodes with 554 edges (Fig. 6A and B). The network contains 7 clusters. As the P value gradually reduced to 1.0E-08, the PgNF-YA09-16 gene remained strongly correlated with PgNF-YA09-08, PgNF-YA09-14, and PgNF-YA09-15. To explore the closeness of the formed networks, we randomly selected 96 transcripts in database A as negative controls and constructed a coexpression network. The results showed ( Fig. 6C and D) that the PgNF-Y transcripts formed 95 nodes and 269 edges compared to the randomly selected transcripts at P ≤ 5.0E-02, and the number of nodes and edges of the unknown transcripts was 0 when the P value gradually decreased to 1.00E-07. Therefore, we determined that the PgNF-Y transcripts form a tighter regulatory network than the negative controls. To further determine the rigor of the regulatory network, we selected two-thirds of the randomly selected PgNF-Y transcripts (65) to construct the regulatory network and Pg NF -YA PgNF-YB05-01  S21  S23  S27  S38  S40  S34  S10  S12  S30  S8  S41  S9  S29  S26  S33  S37  S1  S4  S35  S7  S36  S5 S25 S15 S14 S18 S13 S22 S6 S2 S17 S42 S28 S16 S31 S3 S39 S11 S20 S24 S19 S32 randomly selected another 65 transcripts as the negative control ( Fig. 6E and F). At P ≤ 5.0E-02, the 65 PgNF-Y transcripts formed a regulatory network with 63 nodes, and at P ≤ 1.0E-08, the PgNF-Y transcripts formed 2 nodes and 1 edge; the number of nodes and edges was 0 for the unknown transcripts at P ≤ 1.00E-07. Thus, the PgNF-Y transcripts are more likely to form a coexpression network than the randomly selected transcripts.

Analysis of the response of PgNF-Y genes under salt stress treatment
Ginseng grown in the wild is subject to a variety of factors, and gene expression in a specific environment is often used to predict gene function. To verify the response of PgNF-Y genes to salt stress, we downloaded the known salt tolerance-related NF-Y nucleic acid sequences from the NCBI database and used local BLAST to initially screen three  S9 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21  (Fig. 7). Under salt stress, the expression of the PgNF-YC07-04 gene was significantly increased in ginseng adventitious roots compared with the control. This result further indicates that the PgNF-YC07-04 gene has a strong response to salt stress and plays an important role in the biological process of salt stress resistance in ginseng. This result also provides genetic resources for the study of salt stress resistance and gene breeding in Jilin ginseng.

Discussion
The NF-Y gene family is prevalent in plants. Therefore, the NF-Y transcription factor family has been extensively studied in plants, including Arabidopsis thaliana [27], Glycine max [7], Triticum aestivum [28], and Lotus japonicus [29]. The functional diversity of NF-Y gene  We found that the number of NF-Y gene family members in plants does not seem to differ between herbaceous and woody plants. Polyploidy or whole genome duplication (WGD) is a common phenomenon in angiosperms [30], and the NF-Y transcription factor family in ginseng may have expanded as a result. Genome sequencing has shown that most of the genes specifying core biological functions are common to all eukaryotes [26]. Multiple potential functions exist for PgNF-Y transcripts. Previous studies have shown that NF-Y transcription factors are involved in the regulation of biological processes such as flowering [31], photomorphogenesis [32], and abiotic stress [10] in Arabidopsis by binding to the CCAAT box [33]. According to the GO annotation results, 67 PgNF-Y transcripts were annotated to molecular functions (binding and transcription regulator activity); therefore, these genes are likely to bind the CCAAT box of other genes to regulate the growth and development of Jilin ginseng. Nine PgNF-Y transcripts were annotated to biological processes, and 91 PgNF-Y transcripts were annotated to cellular components, suggesting that PgNF-Y genes act not only as a functional gene but also as structural genes in Jilin ginseng. In summary, the PgNF-Y transcripts are functionally diverse in Jilin ginseng.
Clarifying the expression pattern of PgNF-Y transcripts is helpful for exploring the function of these transcripts. From the analysis of the expression levels of PgNF-Y transcripts in the 42 farm cultivars, the relatively close expression patterns of 115 PgNF-Y transcripts suggest that they are widely expressed in Jilin ginseng. The PgNF-YB13-02 and PgNF-YC08-06 genes had high expression levels in the 42 farm cultivars; therefore, these two genes may be housekeeping genes in Jilin ginseng and thus maintain the basic life activities of the plant. The expression of PgNF-Y genes in 14 different tissues of 4-year-old ginseng showed that their expression was tissue specific, and PgNF-YA12, PgNF-YA13, PgNF-YB01, PgNF-YB08-15, PgNF-YC10, and PgNF-YC12 were expressed only in the fruit pedicel, suggesting that these five PgNF-Y transcripts may be involved in the development of ginseng fruit pedicels. The PgNF-YC13 gene was expressed only in leg roots and may be related to their development in ginseng. The median expression levels of the PgNF-Y gene in the leaf blade and fruit pedicel were higher than that in other tissues, indicating that PgNF-Ys are likely to be involved in ginseng fruit and flower development. In ginseng roots of plants at different ages, approximately 42% of the PgNF-Y transcripts were expressed at different times, and approximately 13% of the PgNF-Y transcripts were expressed only in a specific year. The median expression levels of PgNF-Y genes across the four ages was fairly consistent, indicating that the expression levels of the PgNF-Y gene family members did not increase with growth year. The specific expression of PgNF-Y transcripts at four different ages suggested that not all PgNF-Y transcripts were constitutively expressed in Jilin ginseng, and some transcripts might be induced to be expressed in response to changes in external conditions such as soil and climate.
The NF-Y transcription factor is a heterotrimer formed by evolutionarily conserved subunits: NF-YA, NF-YB, and NF-YC [34]. NF-YB and NF-YC contain a histone folding domain (HFD), and NF-YA binds to the NF-YB/ The expressions analysis of PgNF-YB02, PgNF-YC09, and PgNF-YC07-04 genes in the roots treated with salt stresses using the qRT-PCR. The 2 −△△ . Ct method was used to evaluate the relative expression, and the expression levels of genes in the control were defined as "1". The values are presented as the means of three replicates. "*" as significant at P ≤ 0.05, "**" as significant at P ≤ 0.01 NF-YC dimer [35]. There are 30 predicted NF-Y members in the Arabidopsis genome; in theory, this could result in approximately 1,000 heterotrimeric combinations [36]. In the coexpression network of 42 farm cultivars, we found that PgNF-Y formed some small clusters with close relationships at P ≤ 5.0E-02, suggesting that such heterologous trimers may also be formed in Jilin ginseng. However, this result needs to be verified using yeast two-hybrid and yeast three-hybrid techniques.
The cultivation time for both garden and forest ginseng is usually 5-7 years. During the long cultivation cycle, the soil environment is one of the key factors controlling the quality of ginseng [37]. A recently identified salt tolerance gene, NF-YC13, was found in indica rice [38]; the TaNF-YA10-1 gene was isolated from the salt-tolerant wheat variety SR3; and overexpression of the NF-YA10 gene in Arabidopsis thaliana regulates the response of Arabidopsis to salt stress [21]. In this paper, our fluorescence quantitative PCR results showed that all three transcripts, PgNF-YB02, PgNF-YC09, and PgNF-YC07-04, in ginseng responded to salt stress. This result also indicates that the NF-Y gene family is prevalent in plants responding to salt stress.

Conclusion
In this study, we screened 40 PgNF-Y genes from Jilin ginseng and analyzed their evolution, structure, function, expression pattern and coexpression network to verify the response of PgNF-Y genes to salt stress treatment. The results of the above analysis showed that PgNF-Y gene family members are functionally diverse and exhibit tissue specificity, time specificity, and interspecies differences in their expression patterns. PgNF-Y gene family members act synergistically with each other in the plant. Moreover, the PgNF-Y gene family plays an important role in the plant response to salt stress. This study further demonstrates the role of the NF-Y gene family in the response to salt stress and provides a theoretical basis for the genetic breeding of ginseng.